Melanoma is the sixth most common cancer and accounts for approximately ~5% of all newly diagnosed malignancies. This high morbidity and mortality cancer is largely resistant to available therapies. This is due at least in part, to cancer stem cells (CSC) that are increasingly recognized as the primary source of recurrent and therapeutically resistant disease. Oncolytic viruses are a promising new therapeutic that has the potential to target CSC. Early clinical trials indicate that virotherapy is tolerated well but its efficacy is limited by poor cytolytic activity, insufficient tumor penetration and failures to replicate in quiescent tumor cells. Current efforts seek to improve replication but virotherapy strategies based on the induction of programmed cell death (PCD) are not available. Herpes simplex virus (HSV) is a particularly attractive oncolytic virus because it has a broad host spectrum, its genome does not integrate into the host genome thereby precluding insertion mutagenesis, antiviral drugs are available to safeguard against unfavorable virus replication and the virus evades immunity, allowing re-treatment. The proposed studies follow on our recent findings that an HSV-2 mutant deleted in the HSV-2 gene ICP10PK (delta PK) induces melanoma cell death. The activated Ras signaling pathways in melanoma cells provide for selective delta PK replication but oncolysis is mediated by multiple non-redundant PCD pathways. These pathways include increased calpain and caspase activities, upregulation of the epigenetically silenced gene H11 that has a tumor suppressor-like function in melanoma, enhanced expression of the autophagy-related protein Beclin 1, and activation of the inflammatory caspase-1 (pyroptosis). Delta PK is a particularly promising virotherapy because it elicits Th1 cells, which override the suppressive melanoma milieu and it is tolerated well in humans. The proposed studies seek to better elucidate the relationship between the death pathways induced/activated by delta PK and determine whether it can kill melanoma stem cells. The specific aims are: (i) To elucidate the potential contributions of autophagy and pyroptosis to delta PK induced melanoma oncolysis. Focus will be on the cross-talk between these alternate death pathways and established protease pathways and (ii) To characterize delta PK's potential to infect and kill melanoma stem cells both in vitro and in vivo. The working hypothesis is that PCD induction by delta PK is augmented by autophagy and pyroptosis and delta PK infects and kills melanoma stem cells through one or more of these pathways.